This R21 proposal will investigate the structure-function relationships of human glyoxylate/hydroxypyruvate reductase (GRHPR), a key enzyme in glyoxylate and hydroxypyruvate metabolism. Defects in human GRHPR are present in the rare genetic disease primary hyperoxaluria type 2 (PH2). These mutations ultimately result in the buildup of oxalate and the formation and deposition of urinary tract calcium oxalate kidney stones. An altered GRHPR activity could also contribute to idiopathic stone disease, a common debilitating health problem that impacts daily life and incurs significant health care costs. The historical analysis of GRHPR from other organisms has yielded contradictory evidence for the preference of cofactor, the salt dependence of the reaction, and substrate inhibition. In addition, no structural or biochemical experiments have been reported for human GRHPR. The long-term goals of this research are to characterize the kinetic properties of human GRHPR, to identify the structural features that determine its activity, and to understand how these properties are altered in mutant enzymes causing PH2. The proposed study will (Aim 1) determine the crystal structures of human GRHPR alone and in complex with NADPH and (Aim 2) determine the cofactor and substrate specificity of human GRHPR through the biochemical analysis of wild-type and PH2 mutant enzymes. The structures of human GRHPR will enable the mapping of current and future PH2 variants onto the structure and the prediction of the physiological consequences. The biochemical data will reveal the kinetic parameters associated with the interaction of the enzyme with substrates, cofactors and modulating anions. The cha racterization of human GRHPR will help establish a more precise physiological role for the enzyme and help explain how mutations cause disease. Such studies may ultimately lead to improved treatment strategies for individulas with PH2 and possibly those with idiopathic stone disease. [unreadable] [unreadable] [unreadable]